A number of high molecular weight carbohydrates are polymers of glucose in which the glucose units are joined by either alpha-1,6-glucosidic linkages or alpha-1,4-glucosidic linkages. It is of considerable industrial importance to be able to cleave these linkages thereby breaking the large carbohydrate molecules into smaller molecules which are more useful in various applications. The breaking of the glucosidic linkages is frequently carried out by enzymes which are produced by microorganisms.
One group of enzymes known as alpha-amylases cleave the alpha-1,4-glucosidic linkages. The alpha-amylase enzymes are produced by such organisms as Bacillus licheniformis and Bacillus stearothermophilus. Such enzymes do not cleave the alpha-1,6-glucosidic linkages.
Another class of enzymes, sometimes referred to as glucoamylases, are capable of cleaving both alpha-1,6- and alpha-1,4-glucosidic linkages. These enzymes remove one glucose unit at a time from the nonreducing end of the carbohydrate molecule. While they are capable of hydrolyzing alcha-1,6-glucosidic linkages, they hydrolyze the alpha-1,4-glucosidic linkages much more rapidly.
In the conventional dextrose manufacturing process, starch is hydrolyzed in two stages. In the first step, the starch is liquefied by treatment with an alpha-amylase enzyme at a pH between about 5.5 and 7. The liquefied starch is then saccharified by means of a glucoamylase enzyme operating at a pH between 4 and 5.
When the starch hydrolysis process is carried out at the usual concentration of about thirty percent dry solids, only about ninety-six percent dextrose is formed. One reason why conversion does not proceed appreciably beyond this point is due to the presence of a significant number of oligosaccharides in which at least some of the glucose units are joined by alpha-1,6-bonds. Attempts to obtain greater cleavage of these alpha-1,6-bonds by the addition of increased levels of glucoamylase causes repolymerization of dextrose to oligosaccharides.
Similar problems have arisen when starch is converted to high maltose syrups. Oligosaccharides containing alpha-1,6-bonds between the glucose units are not hydrolyzed by the maltogenic enzymes, resulting in a lower percentage of the desired maltose in the syrup.
In order to overcome these problems, previous workers have suggested adding to the glucoamylase, or other saccharifying enzyme, an enzyme which cleaves the alpha-1,6-linkages. Enzymes described as pullulanases have been used for this purpose. These enzymes are capable of hydrolyzing the alpha-1,6-linkages in the polysaccharide pullulan to give the trisaccharide maltotriose. They do not hydrolyze the alpha-1,4-linkages in pullulan. The first pullulanase described was an enzyme produced by Klebsiella pneumoniae (Aerobacter aerocenes). Reference to its use in a process for hydrolyzing starch is given in U.S. Pat. No. 3,897,305. However, this enzyme has two drawbacks. It is generally active at a pH of 5.5 to 6 where the activity of glucoamylase is dramatically reduced. In addition, the enzyme is thermolabile and cannot be used at temperatures much above 50.degree. C. In commercial operations, it is preferable to carry out the saccharification reactions at 60.degree. C. or higher in order to reduce the risk of microbial contamination of the substrates.
One pullulanase that has been suggested to overcome the foregoing limitations is extracted from rice by a process disclosed in U.S. Pat. No. 4,734,364. Although this enzyme is more thermoduric and aciduric than the enzyme from Klebsiella pneumoniae, it does not retain as much activity as is desired under the normal saccharification reaction conditions. Furthermore, the enzyme is contaminated with other enzymes when first extracted from rice and requires extensive purification before it can be used in the saccharification process.
An additional enzyme which has good thermostability is disclosed in U.S. Pat. No. 4,628,028. This enzyme, derived from Thermoanaerobium brockii, was classified as a pullulanase based on its ability to hydrolyze pullulan to maltotriose. However, it is not suitable for use in the dextrose manufacturing process because it hydrolyzes very few of the alpha-1,6-glucosidic linkages in starch (Coleman, et al., J. Bacteriology, 169, 4302-07 (1987)).
Another pullulanase which was reported to have improved thermoduric and aciduric properties is produced by the microorganism Bacillus acidopullulyticus. This is described in U.S. Pat. No. 4,560,651 and marketed under the trade name PROMOZYME.
We have now discovered a microorganism which produces a pullulanase enzyme that hydrolyzes the alpha-1,6-glucosidic linkages in starch and has even greater thermostability than the one derived from Bacillus acidopullulyticus. Furthermore, it shows good activity and stability at the acidic pH conditions normally employed for the saccharification of starch. For these reasons, it can be used successfully with glucoamylase to give increased yields of dextrose. It may also be used in conjunction with maltogenic enzymes to produce maltose syrups with high maltose contents.